Abstract
Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, compensatory retrieval of membranes and vesicular proteins is essential. We combined capacitance measurements and pH-imaging via pH-sensitive vesicular protein marker (anti-synaptotagmin2-cypHer5E), and compared the retrieval kinetics of membranes and vesicular proteins at the calyx of Held synapse. Membrane and Syt2 were retrieved with a similar time course when slow endocytosis was elicited. When fast endocytosis was elicited, Syt2 was still retrieved together with the membrane, but endocytosed organelle re-acidification was slowed down, which provides strong evidence for two distinct endocytotic pathways. Strikingly, CaM inhibitors or the inhibition of the Ca(2+)-calmodulin-Munc13-1 signaling pathway only impaired the uptake of Syt2 while leaving membrane retrieval intact, indicating different recycling mechanisms for membranes and vesicle proteins. Our data identify a novel mechanism of stimulus- and Ca(2+)-dependent regulation of coordinated endocytosis of synaptic membranes and vesicle proteins.
Highlights
Synaptic transmission relies on neurotransmitter release from presynaptic terminals, which is mediated by exocytotic fusion of synaptic vesicles (SVs) with the plasma membrane
The 200 mm transverse brainstem slices were incubated for 30 min in a high potassium solution (32.5 mM) containing anti-Syt2-cypHer (0.01 mg/ml) to depolarize the terminals and induce exocytosis followed by endocytosis, in which the anti-Syt2-cypHer is internalized (Figure 1B)
Coordinated cycling of vesicle membrane and vesicle proteins is a critical process by which the pool of releasable SVs is repopulated to maintain synaptic transmission over extended periods of time
Summary
Synaptic transmission relies on neurotransmitter release from presynaptic terminals, which is mediated by exocytotic fusion of synaptic vesicles (SVs) with the plasma membrane. Vesicular components are retrieved by endocytosis, and recycled for subsequent rounds of exocytosis (Sudhof, 2004). Two major modes of vesicle fusion have been proposed, kiss-and-run, in which the membrane fusion occurs transiently and vesicle membrane is retrieved by fusion pore closure (Fesce et al, 1994), and full fusion, where the vesicle membrane collapses into the plasma membrane (Cremona and De Camilli, 1997). In the kiss-and-run scenario, the fusing vesicles are thought to largely maintain their identity as regards membrane and protein composition, but it is not clear how membrane and protein retrieval is coordinated when full fusion occurs
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